Structural analysis and functional improvement of plant polysaccharides degrading enzyme from Clostridium thermocellum

• Main Authors: Han-Yu Shie, 謝邯宇
• Other Authors: Po-Huang Liang
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/84386020550022089559

碩士 === 國立臺灣大學 === 生化科學研究所 === 102 === Part1:The glycoside hydrolase 5 (GH5) family contains more than 3000 prokaryotic and eukaryotic enzymes with a large variety of specificities, including endoglucanses, cellobiohydrolases, chitosanases, mannanases and xylanases. Two GH5 enzymes, CtCel5E and TmCel5A, possess different bi-functional activities, cellulase/xylanase and cellulase/mannanase, respectively, although they share sequence homology. The amino acid sequences of these two enzymes are aligned based on SCHEMA and a block was found significantly different in sequence. As revealed by the two protein crystal structures, a flexible loop (without visible electron density) in this block exists in CtCel5A, but not in TmCel5E. The mutant CtCel5E with replacement of this block by the corresponding one in TmCel5A became a tri-functional enzyme with all three cellulase/xylanase/mannanase activities. Through optimization, the best engineered tri-functional enzyme was found to contain loop region replacement plus a F267A mutation. The tri-functional enzyme in combination with three disaccharide-degrading enzymes allowed the complete degradation of mixed artificial substrate into monosaccharides. Moreover, several other amino acids were mutated to test their roles in determining the substrate specificities. Our study provides rationale for engineering a bi-functional enzyme into a tri-functional enzyme, which could be potentially useful for biomass degradation for biofuel production. Part2:Cellulase system is responsible for degrading cellulosic materials, involving three major groups of enzymes, endoglucanases, exoglucanases and β-glucosidases, which cleave at random the internal amorphous area of the cellulose polysaccharide chain, act in a processive manner on the reducing or nonreducing ends to release cellobiose as a major product, and then hydrolyze soluble cellobiose into glucose that can be further fermented to ethanol. For bioethanol industry, seeking novel types of cellulolytic enzymes or engineering the existing enzymes to improve their abilities are actively pursued1. Combining both cellulase and β-glucosidase in the same organism is a good strategy to produce glucose2-3. Dr. Hsiao-Lin Lee et al. fused cellulases from Clostridium thermocellum, a cellulosomal endoglucanase CtCD, with a β-glucosidase CcBG from Clostridium cellulovorans in a single polypeptide chain to efficiently convert cellulosic substrates into glucose without accumulation of cellobiose and improve the thermostability in comparison with the mixture of single CtCD andCcBG enzymes4. In this study, we found that CtCD-CcBG is an oligomer based on AUC analysis and has a resistance core structure against the protease cleavage. Using cross-link, LC-MS/MS and database search, we identified the interaction sites between CtCD andCcBG. Based on the crystal structures of CtCD and CcBg, we established the modeling structure of CtCD-CcBG to realize that two active sites in CtCD-CcBG are closer so that the fused enzyme has higher efficiency to cleave cellulose into glucose. With the knowledges, we could further design better fused protein of cellulase and β-glucosidase with enhanced substrate channeling to increase the yield of glucose for biomass industry.